Mesenchymal stem/stromal cells (MSC) can be isolated from several adult tissues such as bone marrow, adipose, placenta and umbilical cord among others, and are highly promising sources for regenerative medicine. While bone marrow is the most conventional source of MSC, the major limitation in its clinical application is that the concentration of MSC in bone marrow is very low. Subcutaneous adipose tissue is a promising alternative source as it has a high content of MSC, and can be easily obtained by methods such as liposuction or lipectomy.
Adipose tissue can be enzymatically disrupted to yield two main cell populations: mature adipocytes and the stromal vascular fraction (SVF). The SVF is a heterogeneous cell mixture comprising of preadipocytes, mature endothelial cells (EC), endothelial progenitor cells (EPC), vascular smooth muscle cells (SMC), pericytes, mural cells, macrophages, fibroblasts and adipose-derived stem/stromal cells (ASC). The ASC are self-renewing multipotent mesenchymal progenitors that can be easily differentiated into adipocytes, osteoblasts and chondrocytes. Additionally, several investigators have also derived endothelial, myogenic, hepatic and neuronal lineages from ASC under specific inductive conditions. In addition to their plasticity, ASC also secrete bioactive molecules such as immunomodulators and trophic, antiapoptotic, antiscarring, angiogenic, and mitotic factors. Thus, the SVF and ASC from fat tissue have enormous potential in cell-based therapy.
Non-expanded SVF cells are particularly well-suited for autologous cell therapy where clinical doses of the patient's own fat-derived stem cells can be transplanted back with minimal manipulation. SVF cells have been shown to have therapeutic benefit in several preclinical disease models, as well as in clinical trials for indications such as Crohn's disease, graft-versus-host disease, autoimmune and allergic pathologies like multiple sclerosis and inflammatory bowel disease, myocardial infarction, limb ischemia, non-healing chronic wounds, radiation injury, urinary incontinence etc. (Gimble et al. Stem Cell Research & Therapy 2010). They also have huge potential in cosmetic and reconstructive medicine as they have been shown to prolong survival of autologous fat grafts. A clinical study conducted by Yoshimura et. al. (Yoshimura et. al. Aesth Plast Surg, 2008) has demonstrated efficacy of SVF enrichment in fat grafting for breast augmentation. Fat grafting can be applied for post-surgical breast reconstruction, cosmetic breast augmentation, restructuring of facial folds, wrinkle correction and many other soft-tissue defects. Studies in animal models have shown that enrichment of fat grafts with SVF cells promotes engraftment by improving vascularization of the graft, as well as by enhancing turnover of adipocytes, and secretion of anti-apoptotic factors. In fact, the heterogenous composition of the SVF, particularly the high content of endothelial progenitor cells, is ideal for pro-angiogenic cell therapy and vascular repair. Several groups have identified CD34 positive cells in the SVF, capable of stimulating angiogenesis directly or through the release of growth factors such as IGF-1, HGF and VEGF; and SVF cells have been shown to have neo-vasculogenic potential in animal models.
Conventional procedures for isolation of SVF involve manual processing by enzymatic digestion of the lipoaspirate tissue with collagenase, which breaks down the stromal matrix to release the SVF cells. The SVF is then separated from the fat fraction by centrifugation. The conventional manual procedure of isolation has several limitations in the context of clinical application:
Firstly, the fat tissue needs to be transported from the hospital to a GMP-compliant laboratory, wherein storage, handling and transportation of the fat tissue can affect the yield, viability and quality of cells contained in SVF. Further, the time taken for transportation, isolation and delivery of cells is very long.
Secondly, the patient has to undergo more than one sitting at the point of care, and cannot be used in conditions of emergency where the cells are required immediately (eg: for wound healing, burns, myocardial infarction etc.). Further, bench-top open system processing requires rigorous quality control of the therapeutic product.
A few approaches to develop an automated, closed device/system for processing stem cells are already in place. One such automated system for processing of biological samples is disclosed in the PCT publication number WO2005012480 hereinafter referred as '480 publication. The automated system of the '480 publication includes one or more of a collection chamber, a processing chamber, a waste chamber, an output chamber and a sample chamber. The various chambers are coupled together via one or more conduits such that fluids containing biological material may pass from one chamber to another in a closed, sterile fluid/tissue pathway. In certain embodiments, the waste chamber, the output chamber and the sample chamber are optional. The system also includes a plurality of filters. The filters are effective to separate the stem cells and/or progenitor cells from, among other things, collagen, adipocytes, and tissue disaggregation agents that may be present in the solution in connection with the processing of adipose tissue.
Another such automated system for processing of biological samples is disclosed in US Patent publication no. US 20080014181 herein after referred as '181 publication. The automated system of '181 publication apparatus can be used in combination with complementary devices such as cell collection device and/or a sodding apparatus to support various therapies. The automated apparatus of '181 publication includes a cell separation apparatus having a media reservoir. A cell processing device is provided in the cell separation apparatus, wherein the cell processing device comprises at least one inlet and at least one outlet, a first lobe and a second lobe. Further, the cell separation apparatus comprises at least one pump, and at least one valve adapted to divert or prevent fluid flow.
The automated devices disclosed in the prior art documents employs fluid supply mediums such as tubes, hoses for supplying the liquids in between storage containers and the processing container. The fluid supply mediums are to be connected manually before starting the process, which can contribute to errors. Manual connection is also a risk factor that can compromise the sterility or aseptic nature of the cell isolation system. Improper handling can introduce microbial contamination or a breach in the closed nature of the cell isolation system. In addition, to supply the fluid through fluid mediums, pumps are used. The pumps draw the fluid from the storage containers, and supply it to the cell concentration chamber, thereby impart pressure onto the liquid. It is known that the tissues used for extracting cells should not be subjected to pressure, since it will affect the cells in the tissue and thereby reduces the yield of viable cells. Hence there is a need to develop a system for processing biological tissues in aseptic conditions without use of tubes and pumps.
In light of foregoing discussion, it is necessary to develop an improved automated system for processing of biological tissue samples to overcome the problems stated above.